Sanne Hoeks

TH17 within first three months of life 47 4 INTRODUCTION The neonatal immune system has been shown to respond differently when compared with the adult system. Most research on this phenomenon has been performed with cord blood (CB) cells as a representative for the neonatal immune system, showing differences in cytokine production, 1 receptor expression, 2 and cell differentiation capacity. 3 Because of ethical restrictions, it is hard to obtain cells from healthy newborns and infants. Children prenatally diagnosed with an orofacial cleft will have several surgical corrections in the first year of life, providing the opportunity to study immune maturations with longitudinal samples (see Table E1 and methods section). To provide an insight into the changes in the composition of peripheral blood cells after birth, we analyzed the relative fractions of T cells, monocytes, and B cells of all infants at different ages. Although the total number of T cells did not change, the ratio between CD4 and CD8 cells decreased after birth. The number of monocytes gradually decreased over the first year of life but was higher again in adults. B cells showed an inverse pattern (see Fig E1, A-E ). We further characterized subtypes of CD4 + T-helper cells. Most of these cells had a naive (CD45RA + ) phenotype in CB (Fig 1). Although we hypothesized that colonization of the mucosal tissues early after birth would result in a rapid increase in cells with a memory (CD45RO + ) phenotype, we observed only a slight change over the first year of life (Fig 1, A ). No differences were found in the percentages of Forkhead box protein 3 (FOXP3 + ) regulatory T (Treg) cells, but we did observe differences in the ratios of subpopulations as described by Miyara et al. 4 Resting Treg cells in population I are described as potent suppressors that are not in cell cycle. These cells were significantly more abundant throughout the first year of life. Activated Treg cells are not only potent suppressors but also express proliferation markers. These cells were present in equal proportions in all age groups. Population III has been described as consisting of less stable Treg cells with regard to the DNA methylation status of the FOXP3 locus and containing IL-17–producing cells while their suppressive capacity is under debate. 4, 5 These cells were significantly more abundant in adult samples. Next, we assessed the cytokine production of isolated PBMCs after activation by phorbol 12-myristate 13-acetate and ionomycin for 4 hours. Very low concentrations of T-cell cytokines IL-17, IL-13, and IL-10 were found, without age-dependent differences (not shown). IL-1β, IL-6, and TNFα were significantly lower in CB samples as shown before6 and increased with age (Fig 1, C-E ). IFNγ was the only T-cell cytokine observed in considerable amounts in adult samples, but not in CB and 3-month samples (Fig E2). So, the ex vivo phenotype of all infant samples showed a more naive and regulatory phenotype and an increasing capacity of inflammatory cytokine production with age.